The present invention relates to a method for detecting the presence of a nucleic acid sequence of interest, and to a kit of components for performing the assay method.
A number of nucleic acid amplification processes are described in the prior art. One such well known process, polymerase chain reaction (PCR), is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. The PCR process involves the use of nucleic acid primers which anneal to opposite strands of a DNA duplex; these primers are extended using thermostable DNA polymerase in the presence of nucleotide triphosphates to yield two duplex copies of the original nucleic acid sequence. Successive cycles of denaturation, annealing and extension are undertaken in order to further amplify copies of the original nucleic acid sequence.
This method has some disadvantages including the need for adjusting reaction temperatures alternately between intermediate (e.g. 50xc2x0 C.-55xc2x0 C.) and high (e.g. 90xc2x0 C.-95xc2x0 C.) temperatures, involving repeated thermal cycling. Also the time scale required for multiple cycles of large temperature transitions to achieve amplification of a nucleic acid sequence and the occurrence of sequence errors in the amplified copies of the nucleic acid sequence is a major problem, as errors occur during multiple copying of long sequence tracts. Additionally, detection of the amplified nucleic acid sequence generally requires further processes e.g. agarose gel electrophoresis.
Alternative nucleic acid amplification processes are disclosed in WO 88/10315 (Siska Diagnostics) and European patent nos. 329,822 (Cangene) and 373,960 (Siska Diagnostics). These amplification processes are based on a reaction comprising alternate cycles of DNA and RNA synthesis. This alternating RNA/DNA synthesis is achieved principally through the annealing of oligonucleotides adjacent to a specific DNA sequence, whereby the annealed oligonucleotides comprise a transcriptional promoter and initiation site. The RNA copies of the specific sequence so produced, or alternatively an input sample comprising a specific RNA sequence, are then copied as DNA strands using a nucleic acid primer and the RNA from the resulting DNA:RNA hybrid is either removed by denaturation (WO 88/10315) or removed with RNase H (EP 329,882 and EP 373,960). The annealing of oligonucleotides forming a transcription promoter is then repeated in order to repeat RNA production. Amplification is thus achieved principally through the use of efficient RNA polymerases to produce an excess of RNA copies over DNA templates.
The RNase version of this method has great advantages over PCR in that amplification can potentially be achieved at substantially a single temperature (i.e. isothermally). Additionally, a much greater level of amplification per cycle can be achieved than for PCR i.e. a doubling of DNA copies per cycle for PCR, compared with 10-100 RNA copies per cycle using T7 RNA polymerase. A disadvantage associated with the DNA:RNA cycling method described in EP 329,822 is that it requires test nucleic acid with discrete ends for the annealing of oligonucleotides to create the transcriptional promoter. This poses difficulties in detection of, for example, specific genes in long DNA molecules. Further disadvantages of this method are that at least three enzymes are required to undertake the DNA:RNA cycling with potentially deleterious consequences for stability, cost and reproducibility; and that one or more further processes are invariably required (e.g. gel electrophoresis) for detection of the amplified nucleic acid sequence.
The processes described above all refer to methods whereby a specific nucleic acid region is directly copied and these nucleic acid (RNA and/or DNA) copies are further copied to achieve amplification. The variability between various nucleic acid sequences is such that the rates of amplification between different sequences by the same process are likely to differ thus presenting problems for example in quantitating the original amount of specific nucleic acid.
The prior art methods described above have a number of disadvantages in the amplification of their target nucleic acid. It seems to the present inventors that a method for the sensitive detection of a specific target nucleic acid sequence should have the following characteristics:
a) the process should not necessarily require copying of the target sequence;
b) the process should not involve multiple copying of long tracts of sequence to minimise sequence errors;
c) the process should be generally applicable to both DNA and RNA target sequences, including specific sequences without discrete ends;
d) the signal should result from the two or more different hybridisation events so as to improve specificity;
e) the process should include an option for detection of hybridised probe without any additional processes.
A nucleic acid amplification process that fulfils the above desiderata is disclosed in WO 93/06240 (Cytocell Ltd). This amplification process is centred around the use of two nucleic acid probes which contact the target nucleic acid, portions of said probes being capable of hybridising to the sequence of interest such that the probes are adjacent or substantially adjacent to one another, so as to enable other portions of the first and second probes to become annealed to each other. Following annealing, chain extension of one of the probes is achieved by using part of the other probe as a template. Amplification of the extended probe is typically accomplished by: hybridisation of a further probe substantially complementary to part of the newly synthesised sequence of the extended first probe; extending the further probe by use of an appropriate polymerase using the extended first probe as a template; and separating the extended first and further probes, such that the extended further probe can act as a template for the extension of other first probe molecules, and the extended first probe can act as a template for the extension of other further probe molecules.
Other discolosures of interest include U.S. Pat. Nos. 5,451,503 and 5,424,413 (Gen-Probe, Inc.), which refer to the possibility of forming stem/loop structures upon hybridising a nucleic acid probe to a target sequence. The stem/loop may be formed in either the probe or the target sequence. The documents teach the detection of the duplex stem by various means. WO 97/19193 (Yale University, published May 29, 1997) describes a method of amplifying nucleic acid by means of xe2x80x9crolling circle replicationxe2x80x9d, which produces long concatameric copies of circular probe molecules. The preferred method involves the hybridisation of a linear probe molecule to a target sequence of interest, which brines together non-contiguous portions of the probe molecule, which are then joined in a ligation step to form closed circular nucleic acid molecules. A second probe is hybridised to the circular molecule to initiate rolling circle replication.
Detection of nucleic acid target sequences of interest may be useful clinically (e.g. detecting nucleic acid belonging to pathogens, thus aiding diagnosis of infectious disease, or detecting chromosomal abnormalities such as the xe2x80x9cPhiladelphiaxe2x80x9d chromosomal translocation associated with certain cancers), or in public health or environmental fields (e.g. detecting the presence of pathogens such as Salmonella spp in foodstuffs and the like, or detecting E. coli, an indicator of faecal contamination, in water supplies and the like).
It should also be mentioned that those skilled in the art are also acquainted with a substance known as PNA (or peptide nucleic acid). PNA comprises the conventional base compounds present in RNA or DNA. However, instead of the bases being covalently bound to a sugar/phosphate backbone, the bases are joined to a peptide backbone. PNA has many properties in common with RNA and DNA: for example, one can form chimeric duplex molecules by annealing a PNA strand to a strand of RNA or DNA. However, PNA also differs from conventional nucleic acid in a number of respects. In particular, the phosphate groups of DNA carry negative charges, and the repulsion between like charges means that hybridisation of DNA strands must generally be performed in the presence of cations (e.g. aqueous salt solutions) to reduce the electrostatic repulsion between the strands. PNA does not comprise negatively charged phosphate groups and so will generally hybridise more readily than conventional nucleic acid strands.
The present invention typically fulfils all the aforementioned desiderata. In most embodiments, this is achieved by the hybridisation of a long DNA molecule (DNA probe) which xe2x80x98loops outxe2x80x99 non-complementary sequence to the target sequence; a second probe hybridises to the resulting xe2x80x9cloopxe2x80x9d and may be extended. In the absence of target sequence, no loop is formed therefore the second probe cannot hybridise and hence prime the de novo synthesis of nucleic acid. Moreover, the resulting new complementary nucleic acid can be amplified or detected by a range of alternatives including the use of additional DNA probes or enzymes. An overall scheme for the formation of a loop nucleic acid molecule for subsequent detection or amplification is shown in FIG. 1. It will be understood that either the probe or the target nucleic acid could be looped out in the operation of the present invention.
Therefore, in a first aspect the invention provides a method of detecting the presence of a nucleic acid target sequence of interest in a sample, comprising the steps of:
(a) reacting the sample containing the target sequence of interest with a first nucleic acid probe, so as to cause hybridisation between complementary portions of the target and the probe, wherein the probe comprises 5xe2x80x2 and 3xe2x80x2 portions complementary to respective, substantially adjacent portions of the target sequence and an intervening non-complementary portion which does not become hybridised to the target, thereby creating a loop region looped out from a complex formed between the first probe and the target, such that non-contiguous portions of the first probe are brought into close proximity;
(b) hybridising a second nucleic acid probe to the non-contiguous portions of the first probe;
(c) initiating nucleic acid synthesis, using the first probe as template, in a manner dependent upon hybridisation of the second probe to the first probe; and
(d) detecting the newly synthesized nucleic acid from step (c) above.
The method of the invention may be used qualitatively or quantitatively.
Those skilled in the art will appreciate that the first and/or second nucleic acid probes need not consist exclusively of nucleic acid but may comprise e.g. PNA, or labelling reagents. Similarly, the target may comprise RNA or DNA.
Generally the first probe will comprise between 50 and 5,000 nucleotides, preferably between 75 and 1,000 nucleotides. The 5xe2x80x2 and 3xe2x80x2 portions complementary to the target sequence may be of different length (in one embodiment, detailed below, the 5xe2x80x2 complementary portion is much longer than the 3xe2x80x2 complementary portion). Typically each complementary portion will comprise between 16 and 35 nucleotides. The intervening non-complementary portion of the first probe will typically comprise 30-1,000 nucleotides, preferably 50-500 nucleotides.
The second probe will normally be smaller than the first probe, typically comprising 15-100 nucleotides, preferably 15-50 nucleotides.
Those skilled in the art will appreciate that the second probe will hybridise to the intervening portion of the first probe only if the 5xe2x80x2 and 3xe2x80x2 complementary portions of the first probe are themselves hybridised to the target sequence of interest: it is the hybridisation of the 5xe2x80x2 and 3xe2x80x2 complementary portions to the target which brings into close proximity the non-contiguous parts of the intervening portion of the first probe which, in the absence of the target sequence of interest, remain separated from each other. Accordingly, the 5xe2x80x2 and 3xe2x80x2 portions of the first probe must hybridise to respective complementary portions of the target which are substantially adjacent (e.g. within 10 nucleotides, preferably within 5 nucleotides of each other, most preferably contiguous).
The second nucleotide probe is preferably designed such that each region of complementarity to the juxtaposed non-contiguous loop sequences has a melting temperature [Tm] of at least Tm xe2x88x9210xc2x0 C. of the total Tm for the annealing of the whole molecule. Therefore, the assay conditions are preferably designed such that annealing of probes occurs at greater than Tm +10xc2x0 C. (preferably +15xc2x0 C.) above the Tm of the separate complementary regions of the second probe. Hence, hybridisation of the second probe will only occur when sequences in the loop become juxtaposed i.e. only in the presence of target under the assay conditions.
The precise conditions selected for performance of the assay will depend on the length and sequence of the probes and the target. The person skilled in the art will readily be able to vary the conditions if necessary, using trial and error, to obtain appropriate results, with the benefit of the present disclosure.
Once the first and second probes have hybridised to each other, two alternative methods are envisaged for initiating nucleic acid synthesis according to step (c) above.
In one method (the xe2x80x9cprimer extensionxe2x80x9d method), the second probe acts as a primer for de novo nucleic acid synthesis using the first probe as a template. Nucleotides are added to the 3xe2x80x2 end of the second probe in a conventional manner by an RNA or DNA polymerase. Preferably a DNA polymerase is used, to synthesis DNA (although, mutant forms of RNA are known which may synthesise RNA or DNA [see: Kostyuk et al., 1995 FEBS Letts. 369, 165-168]). If desired, the newly synthesised nucleic acid may incorporate labelling reagents (e.g. nucleotides or nucleotide analogues labelled with fluorescent tags, or radiolabels) to facilitate detection of the newly synthesised nucleic acid (in step (d) above).
In embodiments using the xe2x80x9cprimer extensionxe2x80x9d method, it is preferred that the 3xe2x80x2 end of the first probe is blocked, to prevent the first probe being extended in competition with extension of the second probe. Suitable blocking means are well known to those skilled in the art and include, for example, the use of phosphate groups or of nucleotide analogues (e.g. a dideoxynucleotide) at the 3xe2x80x2 end, which prevent extension by polvmerase enzymes.
The nature of the primer extension reaction will largely be determined by the choice of polvmerase. Thus, for example, if a highly prodessive polymerase is employed, one may obtain rolling circle replication of the first probe. Polyinerases suitable for rolling circle replication ideally are capable of displacing the second probe from the first probe template (i.e. have a xe2x80x9cstrand displacementxe2x80x9d activity), which is necessary to obtain multiple copies of the first probe. It is also preferred that such polymerases lack a 5xe2x80x2 to 3xe2x80x2 exonuclease activity which, if present, would tend to cause digestion of the newly-synthesised nucleic acid. In some embodiments of the present invention the template (i.e. the first probe) is not a covalently closed circular nucleic acid molecule (e.g. is a loop, in which the ends are very close together). Accordingly, it is preferred that a highly processive enzyme is employed in such embodiments if rolling circule replication is desired. A particularly preferred polymerase is that obtainable from phage xc3x829 (see U.S. Pat. Nos. 5,198,543 and 5,001,050 and Gutixc3xa9rrez et al, 1991 J. Biol. Chem. 266, 2104-2111). Less preferred are polymerases obtainable from phage M2 (see Matsumoto et al, 1989 Gene 84, 247) or that obtainable from phage xc3x8PRD1 (see Jung et al, 1987 Proc. Natl. Acad. Sci. USA 84, 8287) or polymerases from M13 or xc3x8x 174. In other embodiments, described below, the first probe can be made into a covalently closed molecule and less processive enzymes may be employed and rolling circle replication may still result. Other polymerases which may be suitable are disclosed in WO 97/19193.
It is noted that strand displacement factors (e.g. helicases) may be of assistance in obtaining rolling circle replication, and that such factors may allow an enzyme to perform rolling circle replication which would not normally be caused by such an enzyme in the absence of a strand displacement factor. A number of strand displacement factors are known to those skilled in the art (e.g. calf thymes helicase, disclosed by Siegal et al, 1992 J. Biol. Chem. 267, 13,629). Other strand displacement factors are described in WO 97/19193.
As outlined above, in some embodiments, the first probe can be converted into a covalently closed molecule, which facilitates the performance of rolling cirle replication. In such embodiments the first probe comprises, internal to the 5xe2x80x2 and 3xe2x80x2 portions complementary to the target, two relatively short self-complementary regions, one (the xe2x80x9c5xe2x80x2 self-complementary sequencexe2x80x9d) substantially internally adjacent to the target-complementary 5xe2x80x2 portion, and one (the xe2x80x9c3xe2x80x2 self-complementary sequencexe2x80x9d) substantially internally adjacent to the target-complementary 3xe2x80x2 portion, such that the self-complementary portions flank the intervening non-complementary portion.
When the 5xe2x80x2 and 3xe2x80x2 target-complementary portions hybridise to the target, they bring into close proximity the 5xe2x80x2 and 3xe2x80x2 self-complementary sequences, allowing them to anneal to each other forming a stem structure supporting the looped out non-complementary intervening portion. Each self-complementary sequence typically comprises between 4 and 20 nucleotides, preferably between 6 and 12 nucleotides, such that the stem structure (when formed) comprises a corresponding number of base pairs. Those skilled in the art will appreciate that the self-complementary sequences must be short relative to the 5xe2x80x2 and 3xe2x80x2 target-complementary portions, otherwise the self-complementary sequences may become annealed in the absence of the target sequence, which would in turn allow the second probe to hybridise to the first probe. Accordingly, the region of complementarity producing the stem will preferably be such that the Tm of the stem structure will be less than the overall Tm of the structure stabilised by target nucleic acid. Hence, assay conditions are designed such that the reaction proceeds at least +10xc2x0 C. (preferably +15xc2x0 C.) above the Tm of the stem complementary region.
The double stranded stem structure formed by the annealing of the 5xe2x80x2 and 3xe2x80x2 self-complementary sequences will typically comprise a cleavage site, preferably a restriction endonuclease recognition site. The sequence of the first and second probes will preferably be designed such that the site will be unique to the stem structure. Whilst a restriction endonuclease site is preferred, other specific cleavage sites may be employed (e.g. one recognised by a ribozyme, or by a cleavase). Alternatively, moderately specific cleavage may be obtained by chemical treatment (e.g. with EDTA or other Fe2+ chelator).
Accordingly, treatment of the complex formed between the target, the first probe and the second probe, with the appropriate cleavage mediator (e.g. restriction endonuclease) will cleave the stem structure, releasing the first probe (and any second probe hybridised thereto) from the target sequence. Cleavage of the probe(s) from the target sequence may have the advantage of facilitating subsequent nucleic acid synthesis and/or detection of newly synthesised nucleic acid (e.g. by avoiding steric hindrance resulting from the presence of a possibly very large target sequence). The second probe is desirably selected so as to comprise a short sequence which is complementary to the 5xe2x80x2 and 3xe2x80x2 self-complementary sequences. Accordingly, the second probe is able to hybridise fully to the self-complementary sequences of the first probe once the stem structure has been cleaved by the endonuclease. Thus, a molecule is formed which comprises partially double stranded, nicked nucleic acid. As will be apparent to those skilled in the art, such a structure may be covalently closed by a ligase, yielding covalently closed circular first probe molecule to which is hybridised the second probe. Primer extension (or, more particularly, rolling circle replication) may then proceed as is conventionally known.
In general, once the second probe molecule has been extended, the newly synthesised nucleic acid may be detected by any of a number of techniques already known to those skilled in the art. As described above, the newly synthesised nucleic acid may incorporate labelling reagents. Alternatively, the nucleic acid may be detected and identified by electrophoresis on polyacrylamide or agarose gels (including the use of capillary electrophoresis, as described by Schwartz and Ulfelder [1992 Anal. Chem. 64, 1737]; or by the hybridisation of a complementary labelled nucleic acid probe; or by the binding of some other sequence-specific reagent (e.g. antibody or antibody fragment, or DNA-binding protein, such as a zinc-finger protein. and the like), such as performance of enzyme linked oligonucleotide sorbant assay (ELOSA), described by Berg er al, (1996 Molec. Cellular Probes 10, 7-14); or by fluorescence polarisation (e.g. see xe2x80x9cDetection of Amplified DNA by Fluorescence polarizationxe2x80x9d [1995] in Beacon Fluorescence Polarization Systemxe2x80x94Applications Guide, PanVera Corporation, Madison, USA).
A particularly preferred detection method is the use of a xe2x80x9cmolecule beaconxe2x80x9d, which technique is described by Tyagi and Kramer (1996 Nature Biotechnology 14, 303). In essence, the technique involves the provision of a molecular beacon sequence which is complementary to the sequence to be detected (i.e. the newly synthesised nucleic acid). The molecular beacon comprises at one end a fluorophore, and at the other end a quenching moiety. The molecular beacon also comprises short self-complemertary sequences which, in the absence of the nucleic acid to be detected, remain annealed such that the quenching moiety is in close proximity to the fluorophore and quenches any fluorescence generated by the fluorophore. However, in the presence of a complementary sequence to be detected the molecular beacon is opened out, separating the fluorophore from the quenching moiety and allowing the fluorophore to fluoresce uninhibited. which fluorescence may be detected in the conventional manner. Suitable fluorophores include FAM (FAM is an abbreviation for xe2x80x9cfluorescein addition monomersxe2x80x9d, which refers to 6 carboxy-fluorescein), EDANS (5-[2xe2x80x2-aminoethyl]aminonaphthalene-1-sulfonic acid), and DABCYL (4-[4xe2x80x2-dimethylaminophenylazo]benzoic acid), and a suitable quenching moiety for FAM includes methyl red.
In a preferred embodiment primer extension of the second probe (whether by rolling circle replication or by conventional xe2x80x9clinearxe2x80x9d primer extension) results in the formation of a functional, double-stranded RNA promoter. Thus, the first probe will comprise one strand of an RNA promoter sequence, and the second strand is provided by extension of the second probe. Numerous suitable RNA promoter sequences are known. Accordingly, upon the formation of such an RNA promoter sequence, the corresponding RNA polymerase will synthesise RNA copies of the first probe in the presence of a supply of ribonucleotides. The two preferred RNA polymerases are T7 RNA polymerase and SP6 RNA polymerase, which are both commercially available and have been well characterised. Other RNA polymerases. with known promoter sequences, may also be suitable. An advantage of this embodiment is that the RNA copies may be produced isothermally (i.e. no temperature cycling is required in order to obtain amplification).
It is preferred that extension of the second probe results in the formation of a plurality of RNA promoters. This may be achieved by designing the sequence of the first probe to comprise a plurality of single stranded RNA promoter sequences, such that synthesis of the complementary nucleic acid strand (by extension of the second probe) results in the formation of a plurality of active, double stranded RNA promoters. This has the advantage of allowing for the production of multiple RNA sequences in a single cycle of RNA synthesis, giving increased amplification of the xe2x80x9csignalxe2x80x9d generated by the presence of a single target sequence of interest.
The RNA copies of the first probe may be detected by ally of the known methods outlined previously (e.g. gel electrophoresis, hybridisation to complementary labelled sequences, incorporation of labelled ribonucleotides, etc). Alternatively, the sequence of the first probe may be such that the RNA copies thereof may act as messenger RNA molecules which, upon translation, produce a detectable polypeptide. A particularly suitable polypeptide would be a small activator molecule, which activates an enzyme or enzyme precursor. An example of such an activator molecule is the xcex1 peptide of xcex2-galactosidase, which activates the M15 mutant form of xcex2-galactosidase. The activated enzyme can be used to give an easily readable colourimetric assay, in the presence of a suitable substrate. This type of assay is described in greater detail in WO 93/01313.
In a particular embodiment, rolling circle replication of the first probe is performed by a DNA polymerase, producing a long, concatameric nucleic acid molecule which comprises a plurality of tandem repeats of the sequence complementary to the first probe sequence. Each repeat may comprise one or more single stranded RNA promoters. If the first probe molecule is present in excess (or additional first probe is added after the initial hybridisation to the target sequence) it will hybridise to the newly synthesised complementary strand, thus forming active double stranded RNA promoters, which can be used to produce multiple RNA molecules for detection as described previously.
In some embodiments, the second probe: comprises a short 5xe2x80x2 portion which is not complementary to the first probe, and thus produces a 5xe2x80x2 tail. This has the advantage of facilitating strand displacement, and so assists in extension of the second probe past its original 5xe2x80x2 end. Thus, (using a polymerase without a significant 5xe2x80x2 to 3xe2x80x2 exonuclease activity), there is enhanced likelihood of obtaining rolling circle replication. Alternatively, in other embodiments, displacement of the 5xe2x80x2 end of the original second probe allows extension of nucleic acid synthesis along towards the 5xe2x80x2 end of the first probe, rather than repeated rolling circle replication within the looped out region of the first probe. One may envisage embodiments where, if the 5xe2x80x2 target-complementary portion of the first probe is sufficiently long, and the 3xe2x80x2 target-complementary portion of the first probe is sufficiently short, strand displacement of the 5xe2x80x2 target-complementary portion during extension of the second probe may be sufficient to destabilise entirely the hybridization between the target sequence and the first probe, such that first probe molecule becomes removed from the target sequence. If fresh first probe molecules are available (e.g. by addition of extra molecules, or because of presence in excess ab initio), they may then become hybridised to the target sequence, annealed to by a second probe molecule, and the cycle repeated by extension of the second probe, and so on. This embodiment may be favoured if there are self-complementary portions in the first probe which form a stem, as described previously.
The foregoing generally relates to embodiments of the invention in which the second probe is subjected to primer extension. However, in an other embodiments, an active RNA promoter is formed simply by the hybridisation of the second probe to the first probe. RNA copies of the first probe may then be synthesised and detected as described above.
It will be apparent to those skilled in the art that a substantially equivalent topological arrangement may be obtained by hybridising a probe so as to cause looping out of the target sequence. Such a method is normally less preferable, because if the target sequence is to be looped out one has less flexibility in selection of the sequence of the looped out region, whereas if the first probe is looped out, one can select and design the sequence of the probe for desirable characteristics to a much greater degree.
Thus, in a second aspect the invention provides a method for detecting the presence of a nucleic acid target sequence of interest in a sample, comprising the steps of:
(a) reacting the sample containing the target sequence of interest with a first nucleic acid probe, so as to cause hybridisation between complementary portions of the target and the probe, wherein the target comprises 5xe2x80x2 and 3xe2x80x2 portions complementary to respective, substantially adjacent portions of the probe and an intervening non-complementary portion which does not become hybridised to the probe, thereby creating a loop region looped out from a complex formed between the probe and the target, such that non-contiguous portions of the target are brought into close proximity;
(b) hybridising a second nucleic acid probe to the non-contiguous portions of the target;
(c) initiating nucleic acid synthesis using the target as template, in a manner dependent upon hybridisation of the second probe to the target; and
(d) detecting the newly synthesised nucleic acid from step (c) above.
Those skilled in the art will appreciate that the techniques used to perform the method of the first aspect of the invention will be broadly applicable to the method of the second aspect, defined above. It may be possible to select a portion of the target sequence which comprises a single stranded RNA promoter, which will be complemented by hybridisation and/or extension of the second probe to form an active double stranded promoter. Alternatively primer extension of the second probe may result in the incorporation of labelled nucleotides or nucleotide analogues (e.g. by rolling circle replication).
In a third aspect the invention provides a kit for performing the method of the first aspect, the kit comprising a first nucleic acid probe molecule for hybridisation to the target sequence of interest, a second nucleic acid probe molecule for hybridisation to non-contiguous portions of the first probe molecule which are brought into close proximity upon hybridisation of the first probe to the target, and instructions for use according to the first aspect. The kit will typically further comprise a DNA polyrnerase and/or an RNA polymerase. Further optional components of the kit include: nucleotides (ribonucleotides or deoxyribonucleotides) for synthesis of nucleic acid; detection reagents (e.g. labelling reagents such as labelled nucleotides or nucleotide analogues, molecular beacon sequences); enzyme cofactors; strand displacement factors; reaction buffers (e.g. for performing hybridisation, or nucleic acid synthesis) and the like. The different components of the kit will typically be provided as an aliquot or aliquots within a package.